In some individuals certain drugs can cause an immune reaction which results in thrombocytopenia. A causal relationship between thrombocytopenia and a specific drug cannot be assumed from clinical history either because the patient is taking multiple drugs or because an associated disorder is present which is known to be complicated by thrombocytopenia. The causal agent may be identified by rechallenging the patient with the suspected drug. This approach, however, has potential risks which may make it ethically unacceptable. Hence, an assay technique which detects the drug-dependent antibodies causing the adverse reaction is the preferred diagnostic approach.
Radioimmunoassay techniques have been utilized to detect drug-dependent antibodies. See, for example, Faig, Douglas and Karpatkin, Simon "A Cumulative Experience with a Simplified Solid-Phase Radioimmunoassay for the Detection of Bound Antiplatelet IgG, Serum Auto-, Allo-, and Drug-Dependent Antibodies," J. Am. Soc. Hem., 60, (4): 807-813 (1982).
The technique disclosed involves the deposition of test platelets in wells of a microtiter plate. The platelets are washed and antihuman IgG (rabbit) is added to each platelet-coated microtiter well. After incubation for 1 hr. at room temperature, the wells are washed, treated with .sup.125 I-staphylococcal protein A, and again are incubated for one hour at room temperature. The wells are washed and the radioactivity of each well is then measured and compared to a control. While the technique is effective, it requires the use of radioactive substances with the attendant hazards.
Another technique utilized for the detection of platelet antibodies is an immunoenzymatic assay. Purified rabbit antihuman IgG antibodies are conjugated to beta-galactosidase with meta-maleimidobenzoyl-hydroxysuccinimide ester as a bifunctional reagent and orthonitrophenyl-beta-galactopyranoside as a substrate to evaluate the enzymatic activity of labelled antiglobulin. The tests are carried out in the liquid phase and the antibodies are detected by immunofluorescence, optical density or other suitable measurements. See for example, Borzini, P. et al., "An Immunoenzymatic Assay for the Detection and Quantitation of Platelet Antibodies: The Platelet Beta-Galactosidase Test (PGT)," J. Immuno. Meth., 14, 323-332 (1981).
Electroimmunoassay techniques have been applied to detection of platelet-associated IgG. See for example, Kunicki, Thomas J., et al., "Direct Quantitation of Platelet-Associated IgG by Electroimmunoassay," Blood 60(1): 54-59 (1982). Electrophoresis has been utilized for the study of quinine and quinidine induced thrombocytopenia. See, for example, Christie, Douglas J., and Aster, Richard H., "Drug-Antibody-Platelet Interaction in Quinine- and Quinidine-Induced Thrombocytopenia," J. Clin. Invest., 70(5): 989-999 (1982).
Enzyme-linked immunoabsorbent assay (ELISA) techniques have been applied to the detection of platelet antibodies. See Schiffer, Charles A., and Young, Virginia, "Detection of Platelet Antibodies Using a Micro-Enzyme-Linked Immunosorbent Assay (ELISA)," Blood, 61(2), 311-317 (1983). Lyophilized platelets are used in ELISA tests to detect platelet-associated IgG by a "sandwich" technique utilizing anti-human IgG conjugated to alkaline phosphatase. The results of the tests were quantified with p-nitrophenyl phosphate as the substrate using an automated micro ELISA reader.
Hegde, U. M., et al. have disclosed an ELISA test for detection of platelet associated IgG which comprises coating an antihuman IgG (AIgG) onto polystyrene, binding solublized platelet-associated IgG (PAIgG) to the AIgG, and subsequently measuring the PAIgG by using an enzyme conjugate and p-nitrophenyl phosphate as substrate. See Hegde, U. M., et al. "Enzyme Linked Immunoassay for the Detection of Platelet Associated IgG," Brit. J. Hema., 48, 39-46 (1981).
U.S. Pat. No. 4,016,043 discloses an enzyme immunoassay (EIA) in which antihuman IgG bound to a polystyrene microtiter plate is treated with human serum or plasma. The treated plate is incubated for about 2 hours at 37.degree. C. The wells of the microtiter plate are emptied and washed with a buffer of a pH of about 7.4.
This technique can be used for detection of Hepatitis B surface antigen wherein an antibody is bound to a polystyrene plate which is thereafter treated with serum or plasma. Horseradish peroxidase is added and the material is incubated to complete the reaction. The results are recorded as a colorimetric reading.
U.S. Pat. No. Re 31,006 discloses a similar EIA technique. It discloses as the substrates enzyme catalase, peroxidase, beta-glucuronidase, glucose-oxidase and galactose-oxidase.
U.S. Pat. No. 3,654,090 discloses a method for labeling an antigen or antibody with an enzyme. An example is a conjugate of human chorionic gonadotropin coupled with horseradish peroxidase.